Low-phosphorus whey protein, manufacturing method thereof, low-phosphorus purified whey hydrolysate and manufacturing method thereof

ABSTRACT

A whey protein having a phosphorus content reduced to below 0.15 mg per gram of protein, a manufacturing method thereof, a low-phosphorus hydrolysate highly purified having a low phosphorus content. The method comprises the steps of adjusting pH of the solution containing the whey protein to below 4, and contacting the solution with a cation exchange resin, sequentially contacting the solution with an anion exchange resin, thereby reducing the phosphorus content to below 0.15 mg per gram of protein, and the highly purified low-phosphorus whey protein hydrolysate of the present invention is available by hydrolyzing the above-mentioned low-phosphorus whey protein with proteases.

TECHNICAL FIELD

The present invention relates to a low-phosphorus whey protein, amanufacturing method thereof, a low-phosphorus purified whey hydrolysateand a manufacturing method thereof. More particularly, the presentinvention relates to a low-phosphorus whey protein useful for enrichingvarious foods with nutritive value and protein, an easy and low-costmanufacturing method of the protein, a low-phosphorus purified wheyhydrolysate which is useful as a substitutional substance of protein oramino acid for various food products and drug medicine and as a materialfor cosmetics, and a manufacturing method thereof.

In the following description, the term "protein (or a whey protein)hydrolysate" is defined as a mixture of peptide and free amino acidavailable from a hydrolysate of protein (or a whey protein); and theterm "free amino acid content" is a weight percentage of the free aminoacid content relative to the total amino acid content in the protein (orthe whey protein) hydrolysate.

BACKGROUND ART

Whey is available as a by-product from manufacture of cheese or caseinfrom cow's milk, and protein contained in the whey (hereinaftersometimes referred to as "whey protein") has a high nutritive value. Awhey protein concentrate available by increasing the whey proteincontent has a further higher nutritive value as well as such excellentproperties as a foaming propertiy, a high solubility and a good gelforming property. It is therefore applied in many food products such asdairy products, drink, meat products, sweets and cakes, and bread.Another use thereof is for enriching infant formula with protein.

More recently, utilization of peptide is attracting the generalattention in various areas because of excellent nutritional andphysiological properties including a high digestibility, a lowantigenicity, a low osmic pressure, and a physiological activity ascompared with a mixture of protein and amino acid of the same amino acidcomposition. Application of peptide is therefore studied, in addition tothe conventional utilization in food products, widely in cosmetics anddrug medicine. Enzymatic hydrolysate of whey protein is studied becauseof the suitability for industrial preparation in a large quantity ofpeptide.

Along with the expansion of uses, these whey protein and hydrolysatesthereof are now required to have a unique quality in response toindividual uses. When using whey protein or a hydrolysate thereof as amaterial for medical purposes, various restrictions are imposed on thechemical composition thereof, thus requiring a high-level purity.

It has recently been clarified that over-ingestion of phosporus fromfoods exerted an adverse effect on bone metabolism, and inhibition ofthe amount of ingested phosphorus is now attracting the generalattention. In the medical area, for example, increase in the phosphorusconcentration in blood of a patient subjected to dialytic treatment toremedy renal failure is now known to cause bone growth inhibition andother diseases, and as a result, it is desired to reduce the phosphoruscontent in foods ingested by such a patient. Since there is unavailablean effective therapeutic method without a side effect againsthyperphosphatemia caused by various factors, there is a demand forlow-phosphorus nutritive foods for patients thereof.

Because there is a demand for foods with a reduced phosphorus content asdescribed above, achievement of a reduced phosphorus content in protein,an essential nutrient, is particularly an important task. For example,when aiming at improving the inorganic composition of whey protein, itis the usual practice to desalt whey protein. It is however verydifficult to reduce the phosphorus content. It has actually beenimpossible to manufacture whey protein with a phosphorus content pergram of protein reduced to below 0.15 mg.

The known conventional methods for removing phosphorus contained infoods include: (a) a method of contacting skim milk having a pH adjustedto 5.2 to 8.0 with an anion exchanger (Japanese Patent ProvisionalPublication No. 60-256,342); (2) a method of adding calcium into whey tocause precipitation of free phosphoric acid in the form of calciumphosphate (Japanese Patent Provisional Publication No. 63-91,037); and(c) a method of contacting a liquid food with active alumina (JapanesePatent Provisional Publication No. 2-49,548).

Several of the present inventors developed a method for treating wheyfor the manufacture of low-phosphorus whey protein (Japanese PatentProvisional Publication No. 2-117,366; hereinafter referred to as the"prior application 1"). This method of the prior application 1 comprisesthe steps of concentrating a sweet whey to more than three times by theultrafiltration method, adjusting pH to 3.0 to 4.5, contacting theconcentrate with a cation exchanger to cause adsorption of protein, andcausing elution of the adsorbed protein from the ion exchanger by meansof a solution of a salt.

On the other hand, when using a protein hydrolysate in place of amineacid as a nitrogen component of an intravenous infusion, for example,antigenicity of the protein hydrolysate must previously be eliminated,and the composition of inorganic components including phosphorus issubjected to restrictions by the composition of the intravenous infusionas a whole. In order to prevent coloring of the liquid caused by aminecarbonyl reaction during high-pressure sterilization or preservation inthe manufacturing steps of the intravenous infusion, free amino acid,inorganic substances and a reducing sugar (e.g., lactose) shouldpreferably be not in coexistence in the protein hydrolysate, andfurthermore, there are imposed restrictions also on endotoxin becausethe infusion is administered into blood. In addition, the free aminoacid content should naturally be the lowest possible because it takesthe place of amino acid.

However, when using protein originating from cow's milk as a materialfor manufacturing whey protein hydrolysate, many ingredients to beadjusted or removed, such as inorganic matters, milk sugar and fat, arecontained in the raw materials, and consequently, a highly purified wheyprotein hydrolysate satisfying all the above-mentioned conditions hasnever been available.

The conventionally known protein hydrolysates having properties relatedwith the above-mentioned conditions and methods for manufacturing thehydrolysates include:

(d) a method for manufacturing a low-allergenized whey proteinhydrolysate, which comprises the steps of enzyme-hydrolyzing a wheyprotein at a pH of 6 to 10 by means of a protein hydrolase, heating sameto inactivate the enzyme, and obtaining the low-allergenized wheyprotein hydrolysate having a molecular weight distribution of up to10,000, a main peak within a range of from 1,000 to 5,000, a free aminoacid content of up to 20% (in weight percentage; this shall apply alsohereafter unless otherwise specified), and en antigenicity of up to1/10,000 that of β-lactoglobulin (Japanese Patent ProvisionalPublication No. 4-112,753); and

(e) as peptide product comprising a peptide having a molecular weight ofup to 6,000 daltons and as required an amino acid, and available throughhydrolysis of a whey not containing an allergic substance or lactose,add a method for manufacturing a peptide product, which comprises thestep of enzymatically hydrolyzing a whey protein residue obtained bydialfiltrating a concentrated whey (Japanese Patent ProvisionalApplication No. 63-502,004).

Furthermore, several of the present inventors have previously appliedfor patent for a low molecular-weight peptide composition, whichcomprises a peptide having a molecular weight of up to 1,000 and notexhibiting antigenicity, a free amino acid content of up to 20%, and anaromatic amino acid content of up to 1.0% the total amino acid content;and s method for manufacturing a low molecular-weight peptidecomposition, comprising the steps of hydrolyzing a protein material witha protein hydrolase until until antigenicity is not observed and 90% ofaromatic amino acid contained in the raw material protein become freeamino acid, and collecting the peptide fraction by the gel filtrationmethod (Japanese Patent Provisional Publication No. 2-138,991;hereinafter referred to as the "prior application 2"); a hydrolysate ofmilk protein which is a peptide mixture having a molecular weight of upto 1,000, and comprises free amino acid accounting for more than 90% ofaromatic amino acid, not having antigenicity of milk protein (JapanesePatent Provisional Publication No. 4-26,604; hereinafter referred to asthe "prior application 3"); a fraction of a hydrolysate of a milkprotein which is a peptide mixture having a molecular weight of up to1,000, daltons, and an aromatic amino acid content relative to the totalamino acid content of up to 5%, and not having antigenicity of milkprotein (Japanese Patent Provisional Publication No. 4-26,605;hereinafter referred to as the "prior application 4"); and anoligopeptide mixture available through hydrolysis of a milk proteinhaving a purity of at least 70%, having a molecular weight distributionof up to 2,000 daltons, an antigen residual activity of up to 10⁻⁴ asmeasured by the ELISA (enzyme linked immuno-sorbent assay) method usingantiwhey protein serum, and a free amino acid content of up to 5%relative to the total amino acid content; and a method for manufacturingan oligopeptide mixture, which comprises the steps of dissolving a wheyprotein having a purity of at least 70% by water to a concentration ofup to 10%, adjusting pH of the resulting aqueous solution to a value offrom 7.5 to 10, and enzyme-hydrolyzing same, inactivating the enzyme byheating or removing the enzyme through ultrafiltration (Japanese PatentProvisional Publication No. 4-248,959; hereinafter referred to as the"prior application 5").

In the above-mentioned methods (a), (b) and (c) among the conventionaltechnologies for removing phosphorus in foods, however, it is impossibleto remove phosphorus to a large extent from milk or a liquid food, andthe limit phosphorus content per gram of protein is 40 mg, 10 mg and 6.4mg, respectively. According to the above-mentioned method of the priorapplication 1, it is possible to reduce the phosphorus content to 0.44mg per gram of protein for whey protein, but it is impossible to reducethe phosphorus content per gram of protein to a slight content of up to0.15 mg.

In the conventional technologies, furthermore, while pH of the proteinsolution was adjusted prior to contacting the solution with a cationexchange resin or an anion exchange resin, it was usual that the lowerlimit of pH adjustment was limited by the occurrence of isoelectricprecipitation of protein to be treated. When contacting the raw materialprotein solution with an H⁺ type cation exchange resin, the solutionbecomes acidic under the effect of the decrease in pH, and specificallyadjusting pH of the solution prior to contacting the solution with H⁺type cation exchange resin was not considered at all in any of theabove-mentioned technologies, except for the prior application 1.

In the above-mentioned conventional technologies including (d) and (e)and the prior applications 2 to 5 covering the protein hydrolysates andthe manufacturing methods thereof, some of the conditions such as thelactose content, the molecular weight distribution, the free amino acidcontent and antigenicity were examined, whereas it was impassible toreduce the phosphorus content per gram of protein to a trace amount ofup to 0.15 mg. A protein hydrolysate having a high degree of purity forwhich all the items of the conditions such as the contents of inorganicsubstances, the lactose content, the molecular weight distribution, thefree amino acid content, antigenicity, and the endotoxin content wereconsidered has never been reported to date.

After filing applications for patent for the mentioned priorapplications 1 to 5, the present inventors carried out extensive studieson a method for manufacturing a whey protein having a further reducedphosphorus content. As a result, it was found possible to remarkablyreduce the phosphorus content of a whey protein by contacting the wheyprotein with a cation exchange resin and an anion exchange resin at a pHfurther lower than in the method of the prior application 1. Theycarried out further studies on hydrolysis of a whey protein having a lowphosphorus content available by this method, and found it possible toobtain a highly refined protein hydrolysate having a phosphorus contentfurther lower than that of the protein hydrolysates of theabove-mentioned prior applications 2 to 5 and contents of inorganicsubstances, a lactose content, a molecular weight distribution, a freeamino acid content, antigenicity and an endotoxin content all reduced.The present invention was thus completed.

Disclosure of Invention

The present invention provides a low-phosphorus whey protein having aphosphorus content of up to 0.15 mg per gram of protein.

The present invention provides also a method for manufacturing alow-phosphorus whey protein, which comprises the steps of adjusting pHof a solution containing a whey protein to a value of up to 4, andcontacting the solution with an H⁺ type cation exchange resin andsequentially contacting the solution with an anion exchange resin,thereby reducing the phosphorus content per gram of protein to below0.15 mg.

The present invention provides a low-phosphorus purified whey proteinhydrolysate having the following properties (1) to (6):

(1) the following inorganic substances are contained in the specifiedamounts per gram of protein:

sodium: up to 20 mg,

potassium: up to 20 mg,

magnesium: up to 0.057 mg,

phosphorus: up to 0.15 mg,

calcium: up to 0.227 mg,

chlorine: up to 0.568 mg;

(2) the lactose content is up to 0.5%;

(3) the fraction having a molecular weight of up to 1,200 is at least90%;

(4) the free amino acid content is up to 6%;

(5) the value of antigenicity as measured by the ELISA (enzyme linkedimmuno-sorbent assay) method is up to 10⁻⁶ of that of β-lactoglobulin;

(6) the endotoxin content per gram of the dried material is up to 10 EU.

The present invention provides a method for manufacturing alow-phosphorus purified whey protein hydrolysate, which comprises thesteps of adjusting pH of a solution containing a whey protein to a valueof up to 4; contacting the solution with H⁺ type cation exchange resinand and sequentially contacting with an OH⁻ type anion exchange resin;adjusting pH of the solution to a value of at least 5 and up to 10;removing lactose from the solution through ultrafiltration; adding anenzyme complex comprising two or more enzymes including a proteasederived from an animal and another protease isolated from aBacillus-genus microorganism, or an enzyme complex comprising three ormore enzymes including a protease derived from an animal, anotherprotease isolated from a Bacillus-genus microorganism and furtheranother protease to the solution, in order to cause enzyme-hydrolysis;heating the solution to inactivate enzymes and simultaneously to causeprecipitation of non-reacting protein; and then removing fats and theprecipitation from the solution through ultrafiltration.

In each of the above-mentioned manufacturing methods of the presentinvention, a preferred embodiment is to adjust pH of the solutioncontaining the whey protein to a value of under 3.

According to the present invention, it is possible to very easilymanufacture at a low cost a whey protein having a remarkably lowphosphorus content that has never been achieved and to treat whey in anindustrial scale in a large quantity.

There is provided also a whey protein hydrolysate having very lowcontents of phosphorus, lactose and endotoxin, and excellent innon-antigenicity and absorbency.

BEST MODE FOR CARRYING OUT THE INVENTION

First, the method for manufacturing a low-phosphorus whey protein of thepresent invention comprises the steps of adjusting pH of a solutioncontaining a whey protein, and contacting the solution with an H⁺ typecation exchange resin and sequentially contacting the solution with ananion exchange resin, thereby reducing the phosphorus content to below0.15 mg per gram of protein.

Whey is a remaining liquid after removal of casein produced by adding anacid or rennet to whole milk or skim milk, and contains from about 0.3to 0.7% protein.

The starting material used in the method for manufacturing alow-phosphorus whey protein of the present invention, which is a wheyprotein concentrate having a protein content of at least 70%, may be acommercially available product, or may be a concentrate available byseparating protein from whey by a conventional method, and concentratingthe thus separated protein to a protein content of at least 70%. Thisconcentrate can be manufactured, for example, by a method ofconcentrating protein while eliminating low molecular weight substancesthrough fractionation with ultrafiltration, a method of concentrating byadsorbing protein to a cation exchanger and an anion exchanger, and thencausing elution thereof, or a method of collecting proteinsimultaneously with desalting and lactose-removal by means of a columnfilled with a gel filtration carrier. The phosphorus content in a wheyprotein concentrate varies with the manufacturing method: for currentlycommercially available protein concentrates, the phosphorus content iswithin a range of from 0.4 to 5.0 mg per gram of protein,

This starting material is diluted to prepare a solution having a wheyprotein concentration of about 5 to 20%, and pH is adjusted to a valueof up to 4, or more preferably, under 3 by adding an acid. Acidsapplicable for pH adjustment include hydrochloric acid, citric acid,lactic acid, acetic acid and sulfuric acid. Because pH of the solutioncontaining the whey protein is near the neutral, the isoelectric point(pH of about 5) of protein is passed through. It is therefore possibleto adjust pH to a prescribed value without causing solidification of thewhey protein by previously determining the amount of added acidnecessary for adjusting pH to a desired value, completing addition ofthat prescribed amount within a period of time of from several secondsto one minute, and promptly mixing and stirring the resultant mixture.

Then, the solution containing the whey protein, having a pH adjusted toa value of up to 4 is contacted first with an H⁺ type cation exchangeresin. The applied cation exchange resin may be any of strong acidic andweak acidic resins including such commercially available products as,for example, DIAION SK18 (trademark; made by Mitsubishi ChemicalIndustries, Ltd.), DUOLITE C-26 (trademark; made by Chemical ProcessCompany). AMBERLITE IR-120B (trademark; made by Organo Company), andDOWEX MSC-1 (trademark; made by Dow Chemical Company).

After contact with this H⁺ type cation exchange resin, pH of thesolution containing the whey protein usually takes a value of about 1 to2.5. When contact is caused with the H⁺ type cation exchange resin nearthe neutral value of pH without adjusting pH of the solution containingthe whey protein, as is clear from test examples described later, notonly the effect of reducing the phosphorus content is not observed, butalso the decrease in pH causes solidification by passage of the proteinthrough the isoelectric point (near pH 5), thereby making it impossibleto conduct a continuous ion exchange resin treatment.

Contact between the solution containing the whey protein having anadjusted pH and the cation exchange resin may be accomplished by anyappropriate method such as the batch stirring method or the columncontinuation method. Any method permitting sufficient contact betweenthe solution and the cation exchange resin may be adopted. Whenconducting the method of the present invention in an industrial scale,the column continuation method is preferable for the easy operation.

As to the mixing ratio of the solution containing the whey protein andthe cation exchange resin, varying with the adsorbing ability of the ionexchange resin, the overall exchange capacity (equivalent) of the cationexchange resin must be larger than the total amount (equivalent) ofcations of the solution containing the whey protein: it shouldpreferably be two to five times as large from the point of view of resinutilization efficiency.

Temperature upon contact between the solution containing the wheyprotein and the cation exchange resin may be within a range of from 0°to 80° C. at which no thermal denaturation of the whey protein iscaused, or should more preferably be within a range of from 0° to 10° C.with a view to preventing putrefaction caused by microorganisms. Thecontact time with the solution containing the whey protein may beappropriately selected by taking account of the temperature upon contactand the adopted manner of contact. In the batch stirring method, forexample, contact is caused in a reaction vessel while conductingstirring and mixing or about 0.5 to 3 hours, whereas in the columncontinuation method, this step is accomplished at a velocity of SV=0.01to 20 h⁻¹, or more preferably, SV=2 to 15 h⁻¹.

Then, the solution containing the whey protein after contact with thecation exchange resin is further contacted with an scion exchange resin.The anion exchange resin used here may be any of a strong basic and weakbasic resins, and applicable ones include such commercially availableproducts as DIAION PA318 (trademark; made by Mitsubishi ChemicalIndustries, Ltd.), DUOLITE A-118 (trademark; made by Chemical ProcessCompany), AMBERLITE IRA-411 (trademark; made by Organo Company), andDOWEX MWA-1 (trademark; made by Dow Chemical Company). The opposite ionas the anion exchange resin may be any of OH⁻ type and Cl⁻ type.Preparation into an OH⁻ type one permits desalting of the solution andreduces acidity through the increase in pH. When neutralizing thesolution after treatment, therefore, it is possible to save theconsumption of a neutralizer (alkali agent). The manner of bringing theanion exchange resin into contact, and conditions for the contact arethe same as in the case of the cation exchange resin.

To recover solids of the residual solution in the resins, the resins maybe washed by purified water.

The value of pH of the solution available through the ion exchangetreatment is usually about 1 to 4 and may be neutralized by using asrequired a neutralizer (alkali agent) such as sodium hydroxide orpotassium hydroxide. It is possible to manufacture a desalted andlactose-removed low-phosphorus whey protein by conducting fixed-volumeflowing water diafiltration by means of an ultrafiltration membrane in asolution state of the resultant low-phosphorus whey protein. Thesolution containing the low-phosphorus whey protein thus obtained may bedirectly prepared into a product, or may as required be prepared intopowder by concentrating and drying by a conventional method.

The thus obtained whey protein has much a very low phosphorus content asup to 0.15 mg per gram of protein, and is applicable as a material forfoods with the use of excellent nutritional value, foaming property andemulsifying property. It is suitable also as a material for nutritivefoods for a patient for whom ingestion of phosphorus is limited such asone suffering from hyper-phosphatemia: it is utilizable as a highlyrefined whey protein having a remarkably reduced phosphorus contentwhich is most difficult to eliminate.

Now, the method for manufacturing a low-phosphorus refined whey proteinhydrolysate of the present invention, which comprises the steps ofadjusting pH of a solution containing a whey protein to a value of up to4, contacting the solution with an H⁻ type cation exchange resin andsequentially contacting the solution with an OH⁻ type anion exchangeresin; adjusting pH of the solution to a value of at least 5 and up to9; removing lactose from the solution through ultrafiltration;enzyme-hydrolyzing the solution by adding an enzyme complex comprisingtwo or more enzymes including a protease derived from an animal andanother protease isolated from a Bacillus-genus microorganism, or anenzyme complex comprising three or more enzymes including a proteasederived from an animal, another protease isolated from a Bacillus-genusmicroorganism, and further another pretease; inactivating the enzymes byheating; causing precipitation of non-reacting portion of the protein,and then uItrafiltering the resultant product, thereby removing theprecipitate and fats.

The starting material used in the method for manufacturing alow-phosphorus refined whey protein hydrolysate of the present inventionis the same whey protein concentrate as the starting material used inthe method for manufacturing the above-mentioned low-phosphorus wheyprotein of the present invention.

This starting material is diluted to prepare a solution having a wheyprotein concentration of about 5 to 20%, and pH is adjusted to a valueof up to 4, or more preferably, under 3 by adding an acid. Acidsapplicable for pH adjustment include hydrochloric acid, citric acid,lactic acid, acetic acid and sulfuric acid. Use of hydrochloric acid ispreferable because it does not exert an adverse effect on flavor of thefinal product, and the OH⁻ type anion exchange resin eliminates Cl⁻ ionswhich do not finally remain. Because pH of the solution containing thewhey protein is near the neutral, the isoelectric point (pH of about 5)of protein is passed through. It is therefore possible to adjust pH to aprescribed value without causing solidification of the whey protein bypreviously determining the amount of added acid necessary for adjustingpH to a desired value, completing addition of that prescribed amountwithin a period of time of from several seconds to one minute, andpromptly mixing and stirring the resultant mixture.

Then, the solution containing the whey protein having a pH adjusted to avalue of up to 4 is contacted first with an H⁺ type cation exchangeresin, and then with an OH⁻ type anion exchange resin for desalting. Thekind of the ion exchange resin selected, the manner of contacting theion exchange resin, and the conditions for contact are the same as inthe above-mentioned method for manufacturing the low-phosphorus wheyprotein of the present invention. In order to keep the contents ofinorganic substances in the finally available whey protein hydrolysate,the solution can be desalted by using an opposite ion of the anionexchange resin prepared in the form of OH⁻ type. Furthermore, since theincreased pH reduces acidity, it is possible, when pH of the solution ismade neutral or alkaline after the ion exchange treatment, to save theconsumption of the neutralizer (alkali agent). To recover solids of theresidual solution in the resins, the resins may be washed by purifiedwater.

Because the desalted whey protein solution thus obtained has an acidicpH, a neutralizer (alkali agent) is added to adjust pH to a value of atleast 5 and up to 10, or more preferably, to a value of at least 6 andup to 9. Neutralizers (alkali agents) applicable for adjustment of pHinclude sodium hydrochloride and potassium hydrochloride. This pHadjustment operation has an object to prevent corrosion by oxidation of,for example, manufacturing facilities, and prevent elution of inorganicions from metallic portions of the manufacturing facilities. Anotherobject is to cause pH of the whey protein solution to match with theoptimum pH range of the enzyme used in the enzyme hydrolysis in thepresent invention. When there is no risk of corrosion by oxidation ofmanufacturing facilities, therefore, it is not necessary to conduct pHadjustment operation of the desalted whey protein solution immediatelyafter the above-mentioned desalting operation with the aim of complyingwith the optimum pH range of enzyme, but is may appropriately be carriedout before enzyme-hydrolysis operation. For example, when there is nocorrosion by oxidation of manufacturing facilities, it is possible toadjust pH of the desalted and lactose-removed whey protein solution tothe optimum pH range of enzyme used in the present invention,immediately before the enzyme-hydrolysis operation after the lactoseremoving operation, the next step, without adjusting pH immediatelyafter the desalting operation. Another possible embodiment comprisesroughly adjusting pH of the above-mentioned desalted whey proteinsolution, immediately after the desalting operation, within a rangepermitting prevention of corrosion by oxidation of manufacturingfacilities, and after conducting the next lactose removing operation,adjusting pH of the desalted and lactose-removed whey protein solutionwithin the optimum pH range of the enzyme used in the present invention,immediately before the enzyme-hydrolysis operation.

Then, lactose contained in the desalted whey protein solution is removedby ultrafiltration. Ultrafiltration membranes having a fractionmolecular weight within a range of from 2,000 to 10,000 are applicable,and any of the ultrafiltration methods common in this technical field isapplicable. Applicable ultrafiltration modules include, for example, theflat membrane type, the tubular type, the spiral type, and the hollowfibre type. When taking account of the separating efficiency andeconomic merits, use of the tubular type or the hollow fibre type ispreferable.

Because β-lactoglobulin and α-lactalbumin in the whey protein containedin the desalted whey protein solution have a molecular weight of about18,000 and about 14,000, respectively, these whey proteins do notpermeate through the ultrafiltration membrane upon ultrafiltration ofthe desalted whey protein solution, but lactose having a smallermolecular weight is discharged as a membrane permeating fraction,Furthermore, lactose can be eliminated almost completely by conductingfixed-volume flowing water diafiltration with purified water. Since thewhey protein does not permeate the ultrafiltration membrane but is heldwithin the membrane, the operation of fixed-volume flowing waterdiafiltration exerts no adverse effect on yield. Because ultrafiltrationcauses inorganic substances to be discharged on the membrane permeationliquid side, desalting effect is also available.

The whey protein concentration of this desalted and lactose-removed wheyprotein solution is adjusted to under 10%, and then an enzyme is addedto the solution.

The enzyme used here is an enzyme complex comprising two or more enzymesincluding a protease derived from an animal and a protease isolated froma Bacillus-genus microorganism, or an enzyme complex comprising three ormore enzymes including a protease derived from an animal, anotherprotease isolated from a Bacillus-genus microorganism, and furtheranother protease, Applicable professes originating from animals includetrypsin, chymotrypsin, and pancreatin, all of which are commerciallyavailable (for example, "PTN 6.0S," a trademark; made by Novo NordiskCompany). Proteases isolated from Bacillus-genus microorganisms includePROTEASE N (trademark; made by Amano Seiyaku Company), BIO-PRASE(trademark; made by Nagase Seikagaku Kogyo Company), PROLEATHER(trademark; made by Amano Seiyaku Company), and ALCALASE (made by NovoNordisk Company).

With a view to reducing antigenicity of the resultant whey proteinhydrolysate, the object is well achieved With an enzume complexcomprising a simple combination of a protease derived from an animal anda protease isolate from a Bacillus-genus microorganism. However, when awhey protein hydrolysate available by the use of an enzyme complex ofsuch a combination is poor in flavor, It is possible to improve flavorby simultaneously using another protease. Such other proteasesapplicsble in this case include PAPAIN, BROMELINE (made by Amano SeiyakuCompany), a protease isolated from an Aspergillus-genus microorganism,and a protease isolated from Penicillium-genus microorganism.

The amount of enzymes used should be within a range of from 3,800 to20,000 activity units per gram of whey protein, and the enzyme complexis added by mixing or dividing.

Because the optimum value of pH of the enzyme used in the presentinvention is within a range of from neutral toward alkali side, thevalue of pH upon hydrolysis should be within a range of at least 5 andup to 10, or more preferably, at least 6 and up to 9.

There is no particular limitation on temperature conditions forhydrolysis based on enzymatic reaction: temperature may be selectedwithin a practicable range including the optimum temperature range inwhich the enzyme action can manifest, and should be within a range of atleast 30° C. and up to 70° C. in general, or more preferably, of atleast 30° C. and up to 60° C., or further more preferably, of at least50° C. and up to 60° C. Particularly, it is possible to preventputrefaction of the whey protein solution during enzymatic reaction bykeeping temperature within a range of at least 50° C. and up to 60° C.

The time required for enzymatic reaction may be determined in advancethrough a preliminary experiment. More specifically, the determinationof time for enzymatic reaction is accomplished, for example, by samplingreaction liquid little by little at certain time intervals from thestart of enzymatic reaction, subjecting the sampled reaction liquid toan arresting treatment of the enzymatic reaction and the ultrafiltrationtreatment of the present invention, drying the resultant filtrate by theconventional method into powder, determining, for this powder, themolecular weight distribution, the free amino acid content andantigenicity by the method described later, and using the enzymaticreaction time in the case where a powder of a desired composition isachieved as the enzymatic reaction time upon executing the presentinvention thereafter. For example, an enzymatic reaction time of from 8to 36 hours is required for obtaining a whey protein hydrolysate havingthe following properties in the present invention:

(i) a fraction having a molecular weight of up to 1,200 accounting forat least 90%;

(ii) a free amino acid content of up to 6%;

(iii) an antigenicity, as measured by the enzyme linked immuno-sorbentassay, of 10⁻⁶ of antigenicity of β-lactoglobulin.

After the stage at which the whey protein hydrolysate has come to havethe above-mentioned properties along with the progress of the enzymaticreaction, the enzyme is inactivated by heating. Inactivation of theenzyme may be accomplished by heating the reaction liquid at atemperature of at least 80° C. for more than six minutes. This heatingcauses generation of an undissolved product of about 20 (vol.) % whencentrifugally separating the reaction liquid.

The whey protein hydrolysate solution after heating and inactivation ofthe enzyme is ultrafiltered to eliminate the undissolved product andfats for purification of the solution and for removal of endotoxin. Theundissolved product, fats and endotoxin do not permeate theultrafiltration membrane but remain on the membrane holding liquid side.It is therefore possible to purify the whey protein hydrolysate solutionand remove endotoxin by collecting the liquid having permeated throughthe membrane. An ultrafiltration membrane having a fractional molecularweight of up to 5,000 is applicable and the commonly adopted method inthis field of art may be applied for ultrafiltration. Applicable modulesfor ultrafiltration include, for example, the flat membrane type, thetubular type, the spiral type and the hollow fibre type. Whenconsidering the separating efficiency and economic merits, use of thetubular type or the hollow fibre type is preferable.

The recovery ratio of peptide which is a valuable solid in the originalliquid can be improved by carrying out fixed-volume flowing waterdiafiltration with purified water.

The resultant liquid may directly be used as a product, or may asrequired by converted into a powder by concentrating and dried by theconventional methods.

The thus obtained low-phosphorus purified whey protein hydrolysate ofthe present invention has the following properties (1) to (6):

(1) containing the following inorganic ingredients in the amounts shownper gram of protein:

sodium: up to 20 mg,

potassium: up to 20 mg,

magnesium: up to 0.057 mg,

phosphorus: up to 0.15 mg,

calcium: up to 0.227 mg,

chlorine: up to 0.568 mg;

(2) a lactose content of up to 0.5%;

(3) a fraction, having a molecular weight of up to 1,200, of at least90%;

(4) a free amine acid content of up to 6%;

(5) an antigenicity, as measured by the enzyme linked immuno-sorbentassay, of up to 10⁻⁶ of antigenicity of β-lactoglobulin;

(6) an amount of endotoxin of up to 10 EU per gram of dried product.

More specifically, while properties of the low-phosphorus purified wheyprotein hydrolysate of the present invention may, for example, be withinthe following ranges (a) to (f), the present invention is not limited tothose ranges:

(a) contents of inorganic ingredients per gram of protein:

sodium: from 0.04 to 17 mg,

potassium: from 0.01 to 17 mg,

magnesium: from 0.03 to 0,05 mg,

phosphorus: from 0.11 to 0.13 mg,

calcium: from 0.15 to 0.20 mg,

chlorine: from 0.40 to 0.50 mg;

(b) a lactose content within a range of from 0.1 to 0.4%;

(c) a fraction having a molecular weight of up to 1,200 within a rangefrom 90 to 94%;

(d) a free amine acid content of from 4 to 6%;

(e) an antigenicity, as measured by the enzyme linked immuno-sorbentassay, of up to 10⁻⁶ of antigenicity of β-lactoglobulin (detection limitof the ELISA method described later);

(f) an amount of endotoxin of from 2 to 8 EU per gram of dried product.

As shown in the above-mentioned properties (1) to (6), thelow-phosphorus purified whey protein hydrolysate is suitably applicableas a nitrogen ingredient of an intravenous infusion, for example, inplace of amino acid because of the low inorganic contents and becauseendotoxin and antigenicity are almost completely eliminated. It ispossible, in this case, to prevent coloring of the liquid caused by theamino carbonyl reaction during high-pressure vapor sterilization in themanufacturing process of an intravenous infusion or during storagethereof, since the contents of Tree amino acid, inorganic ingredientsand lactose are limited to low levels. The low-phosphorus purified wheyprotein hydrolysate is a mixture of peptide and free amino acid. Becauseof the low free amine acid content of up to 6%, an intravenous infusionusing the low-phosphorus puriied whey protein hydrolysate as a nitrogeningredient can be prepared into an infusion of a low osmotic pressure ascompared with an intravenous infusion using an amino acid mixture of thesame chemical composition as a nitrogen ingredient. In addition, sincephosphorus the most difficult to remove among inorganic matters isremarkably reduced, it is applicable as a substitute for proteinexcellent in digestibility having a high degree of refining to be usedas a material for a nutritional meal for a patient of a limitedingestion of phosphorus such as a patient suffering fromhyper-phosphatemia.

The low-phosphorus purified whey protein hydrolysate of the presentinvention was subjected to the following tests:

(1) Measurement of the Inorganic Matter Contents

The contents of sodium, potassium, magnesium, phosphorus and calciumwere determined by the conventional method (edited by the Japan Societyof Analytical Chemistry, Machine Analysis Practice Series, "ICP EmissionAnalysis Method," p. 225, Kyoritsu Shuppan, 1988) per gram of protein ina sample, together with the protein content in a sample measured by theconventional method. The chlorine content was measured by thepotentiometric titration method (Japan Food Industry Association, editedby the Food Analysis Editing Committee, "Food Analysis Methods," 2nded., p. 368, Korin Publishing Company, 1984).

(2) Measurement of Lactose Content

The lactose content was measured by High Performance liquidchromatography (Journal of the Japan Food Industry Association, Vol. 27,No. 7, p. 36, 1980). Using Shodex DC613 (made by Showa Denko Company),elution was caused by means of an eluate having an acetonitrile:waterratio of 75:25 at an elution rate of 1.2 ml/minute. Detection wascarried out by the post-label method Bunseki Kagaku, Section E, Vol. 32,No. 6, p. E207, 1983! by means of a fluorescent detector (made byShimazu Works; SHIMAZU RF530). The lactose content was calculated by theinternal standard method (Japan Society of Analytical Chemistry, editedby Kanto Branch, "High Performance Liquid Chromatography Handbook," 277,Maruzen Company, 1985).

(3) Measurement of Molecular Weight Distribution

The molecular weight distribution was measured by High Performanceliquid chromatography (N. Ui, et al., "High Performance LiquidChromatography of Protein and Peptide," Kogaku, Additional Issue No.102, p. 241, Kagaku Dojin Company, 1984). Elution was caused by means ofa poly hydroxyethyl aspartamide (made by Poly LC Company) column, with50 mM formic acid at an elution rate of 0.5 ml/minute. An RI detector(made by Shimazu Works) was used for detection, and a GPC analysissystem (made by Shimazu Works) was used for data analysis.

(4) Measurement of Free Amino Acid Content

The content of each of the amino acids other than tryptophane, cysteineand methionine was determined by hydrolyzing a sample with 6Nhydrochloric acid at 110° C. for 24 hours, alkaline-decomposing thesample, for tryptophane, with barium hydroxide at 110° C. for 22 hours,or hydrolyzing the sample, for cysteine and methionine, with 6Nhydrochloric acid at 110° C. for 18 hours after a performic acidtreatment, and decomposing same by an appropriate amino acid analyzer(made by Hitachi Seisakusho; Model 835). The free amino acid content wasanalyzed by means of an amino acid analyzer (made by Hitachi Seisakusho;Model 835) and was expressed in percentage of free amino acid relativeto the total content of the individual amino acids as derived from theabove-mentioned analysis of amino acid composition.

(5) Measurement of Antigenicity

Antigenicity was determined by the ELISA (enzyme linked immuno-sorbentassay) method as follows:

Antigenicity was measured by coating a 96-hole plate (made by NunkCompany) with β-lactoglobulin, then after washing, supplying a mixedsolution of rabbit antiserum prepared through sensitization ofβ-lactoglobulin and a sample whey protein hydrolysate to the holes ofthe plate to cause a reaction, then after washing, causing a reaction ofalkali-phosphatase label goat anti-rabbit IgG antibody (made by ZymedLaboratories), then after washing, adding p-nitrophenyl sodium phosphatewhich is an enzyme substrate, adding sodium phosphate, adding 5N sodiumhydroxide after the lapse of 30 minutes to arrest the reaction, andmeasuring the resultant reaction product with a micro-plate reader(Journal of the Japan Infant Allergy Association, vol. 1, No. 2, p. 36,1987).

(6) Measurement of Endotoxin Content

Endotoxin content was measured in accordance with the LIMULUS test (N.Niwa, Journal of the Japan Bacteriology Society, Vol. 30, p, 439, 1975),by means of a Limunlus HSII TESTWAKO (made by Wako Jun-Yaku KogyoCompany), to measure the gel forming time with a toxiometer ET201 (madeby Wako Jun-Yaku Kogyo Company).

Now, the present invention is described further in detail by means ofTESTs.

TEST 1

This test was carried out to investigate the effect of pH of a solutioncontaining a whey protein on the decrease in the phosphorus content.

1) Preparation of Samples

A whey protein concentrate (made by Mirei Company, Germany; a proteincontent of 90% and a phosphorus content of 0.40 mg/gram of protein) wasadded to purified water, to adjust the concentration of the whey proteinto 10%. Samples each weighing 600 g were prepared while adjusting pH bynull (pH of 7.18; Samples 1 and 2), to 4.00 with 3N hydrochloric acid(Samples 3 to 6), to 3.00 (Sample 4) and to 2.00 (Sample 5).

2) Procedures

1. Method 1

Sample 1 was not contacted with an ion exchange resin, but directlysubjected to measurement of the phosphorus content.

2. Method 2

Each of Samples 2 to 5 was passed through a column filled with an H⁺type cation exchange resin AMBERLITE IR-120B (made by Organo Company) inan amount of 50 ml at a velocity of SV=5 h¹ to contact with each other,and then passed through another column filled with an OH⁻ type anionexchange resin AMBERLITE IR-411 (made by Organo Company) in an amount of100 ml to contact with each other, thereby removing phosphorus in thesample.

3. Method 3

Sample 6 was treated in the same manner as in the above-mentioned method2 except that the sample was not contacted with the OH⁻ type anionexchange resin,

4. Measurement of Phosphorus Content

The phosphorus content was measured by the procedres as described abovein the six Samples obtained by the above-mentioned three methods. Thephosphorus content per gram of protein in the sample was calculated onthe basis of the protein content in the sample as measured by theconventional method to test the status of phosphorus removal.

3) Results

The test gave results as shown in Table 1. As is clear from Table 1, inSamples 1 and 2 not subjected to pH adjustment, the ion exchangetreatment reduces the phosphorus content from 0.40 mg only to 0.24 mgper gram of protein. In Samples 3 to 5 which were subjected to a cationexchange resin treatment and an anion exchange resin treatment after pHadjustment to below 4, in contrast, the phosphorus content was reducedto below 0.15 mg per gram of protein in all cases. Also in the casewhere pH of the solution containing the whey protein was adjusted to 4,the phosphorus content in Sample 6 brought into contact only with the H⁺type cation exchange resin was almost the same as that in Sample 2subjected to an ion exchange resin treatment without adjusting pH.

To judge from these results, it is essential to adjust pH of thesolution containing the whey protein to below 4, or more preferably, tobelow 3 prior to contacting the solution with the cation exchange resinand the anion exchange resin. Tests carried out by changing the kind ofwhey protein concentrate and the kind of resin gave almost the sameresults.

                  TABLE 1                                                         ______________________________________                                                           Ion exchange                                                                            Phosphorus content                               Sample No.                                                                              pH       treatment (mg/gram of protein)                             ______________________________________                                        1         7.18*    Not treated                                                                             0.393                                            2         7.18*    H.sup.+, OH.sup.-                                                                       0.241                                            3         4.00     H.sup.+, OH.sup.-                                                                       0.114                                            4         3.00     H.sup.+, OH.sup.-                                                                       0.107                                            5         2.00     H.sup.+, OH.sup.-                                                                       0.093                                            6         4.00     H.sup.+   0.291                                            ______________________________________                                         *Not adjusted                                                            

TEST 2

This test was carried out to investigate the effect of a change in thesequence of anion and cation exchange resins with which the solution isbrought into contact on removal of phosphorus.

1) Preparation of Samples

A whey protein concentrate (made by Calpro Company; a protein content of80% and a phosphorus content of 3.5 mg/gram of protein) was added topurified water to adjust the whey protein concentration to 10%, therebypreparing Samples 7 to 9 with a pH adjusted to 3.00 with 3N hydrochloricacid each in an amount of 100 g.

2) Procedures

(1) Method 1

Sample 7 was not contacted with an ion exchange resin, but directlysubjected to measurement of the phosphorus content.

(2) Method 2

Sample 8 was passed through a column filled with an H⁺ type cationexchange resin AMBERLITE IR-120B (made by Organo Company) in an amountof 18.5 ml at a velocity of SV=5 h⁻¹ to contact with each other, andthen passed through another column filled with a Cl⁻ -type anionexchange resin AMBERLITE IRA-411 (made by Organo Company) in an amountof 41.4 ml at a velocity of SV=5 h¹ to bring them into contact with eachother, thereby removing phosphorus in the sample.

(3) Method 3

Sample 9 was treated in the same manner as in the method 2 except thatthe sample was first contacted with an anion exchange resin.

(4) Measurement of Phosphorus Content

The phosphorus content in the three samples obtained by theabove-mentioned methods was measured in the same manner as in the TEST 1to test the status of phosphorus removal.

3) Results

The results of this test are shown in Table 2. As is clear from Table 2,phosphorus cannot be removed unless the solution is first contacted withthe cation exchange resin regarding the sequence of contact of thesolution containing the whey protein with the cation exchange resin andthe anion exchange resin. In the method of the present invention,therefore, it is essential to bring the solution containing the wheyprotein into contact first with the cation exchange resin, and then withthe anion exchange resin. Tests carried out by changing the kind of wheyprotein concentrate and resin gave almost the same results.

                  TABLE 2                                                         ______________________________________                                                           Ion exchange                                                                            Phosphorus content                               Sample No.                                                                              pH       treatment (mg/gram of protein)                             ______________________________________                                        7         3.00     Not treated                                                                             3.513                                            8         3.00     H.sup.+, OH.sup.-                                                                       0.126                                            9         3.00     OH.sup.-, H'                                                                            3.025                                            ______________________________________                                    

EXAMPLES

Now, the present invention is described further in detail by means ofEXAMPLES. The present invention is not however limited by them.

In the following EXAMPLES, the contents of sodium, potassium, magnesium,phosphorus, calcium and chlorine are expressed in units of mg/gram ofprotein.

In the EXAMPLES of the present invention, the inorganic matter content,the lactose content, the molecular weight distribution, the free aminoacid content, antigenicity and the endotoxin content were measured bythe above-mentioned procedures of TESTS.

Example 1

A whey protein concentrate (made by Mirei Company, Germany; containing90.3% protein, 5.1% sodium, 0.26% potassium, 0.33% magnesium, 0.39%phosphorus and 3.98% calcium) was added to purified water to adjust theconcentration of the whey protein to 10%. Then, 3N hydrochloric acid inan amount of 134 ml was added to 1 kg this solution and pH was adjustedto 3.0. The solution was passed through a column filled with an H⁺ typecation exchange resin AMBERLITE IR-120B (made by Organo Company) in anamount of 75 ml at SV=12.5 h¹ to contact with each other, and thenpassed through another column filled with a Cl- type anion exchangeresin AMBERLITE IRA-411 (made by Organo Company) in an amount of 120 mlat SV=12.5 h¹ to contact with each other. Then, the columns filled withresin were washed by purified water to recover solids of the residualsolution in them. The resultant solution of a pH of 2.11 containing awhey protein in an amount of about 3 kg was recovered, and freeze-driedby the conventional method, thereby obtaining a low-phosphorus wheyprotein powder in an amount of about 96 g.

The thus obtained powder was tested in accordance with theabove-mentioned test methods: the inorganic composition comprised 0.3%sodium, 0.008% potassium, 0.0005% magnesium, 0.11% phosphorus and 0.008%calcium, suggesting that phosphorus was remarkably eliminated.

Example 2

A whey protein concentrate (made by Calpron Company; containing 83.0%protein, 1.45% sodium, 4.0% potassium, 0.65% magnesium, 3.39% calcium)was added to purified water to adjust the concentration of the wheyprotein to 10%. Then, 5N hydrochloric acid in an amount of 76.2 ml wasadded to 1 kg this solution and pH was adjusted to 2.8. The solution waspassed through a column filled with an H⁺ type cation exchange resinAMBERLITE IR-120B (made by Organo Company) in an amount of 100 ml atSV=2.5 h⁻¹ to contact with each other, and then passed through anothercolumn filled with a Cl⁻ type anion exchange resin AMBERLITE IRA-411(made by Organo Company) in an amount of 220 ml at SV=2.5 h¹ to contactwith each other. Then, the columns filled with resins were washed bypurified water to recover solids of the residual solution in them. Theresultant solution of a pH of 1.96 containing a whey protein in anamount of about 3 kg was recovered, and freeze-dried by the conventionalmethod, thereby obtaining a low-phosphorus whey protein powder in anamount of about 84 g.

The thus obtained powder was tested in accordance with theabove-mentioned test methods: the inorganic composition comprised 0.038%sodium, 0.059% potassium, 0.0025% magnesium, 0.125% phosphorus and0.0213% calcium, suggesting that phosphorus was remarkably eliminated.

Example 3

A whey protein concentrate (made by Mirei Company, Germany; containing90.3% protein, 7.7% sodium, 0.60% potassium, 0.4% magnesium, 0.38%phosphorus, and 4.43% calcium) was added to purified water to adjust theconcentration of the whey protein to 12.4%. Then 35% hydrochloric acidin an amount of 68 kg was added to 4.030 kg this solution and pH wasadjusted to 3.05. The solution was passed through a column filled withan H⁺ type cation exchange resin AMBERLITE IR-120B (made by OrganoCompany) in an amount of 350 l at SV=10 h¹ to contact with each other,and then passed through another column filled with an OH⁻ type anionexchange resin AMBERLITE IRA-411 (made by Organo Company) in an amountof 700 l at SV=5 h¹ to contact with each other. Then, the columns filledwith resins were washed by purified water to recover solids of theresidual solution in them. The resultant solution of a pH of 3.50containing a whey protein in an amount of about 6.825 kg was recovered,and freeze-dried by the conventional method, thereby obtaining alow-phosphorus whey protein powder in an amount of about 437 kg.

The thus obtained powder was tested in accordance with theabove-mentioned test methods: the inorganic composition comprised 0.06%sodium, 0.03% potassium, 0.006% magnesium, 0.119% phosphorus and 0.023%calcium, suggesting that phosphorus was remarkably eliminated.

Example 4

A whey protein concentrate (made by Mirei Company, Germany; containing90.3% protein, 5.1% sodium, 0.256% potassium, 0.331% magnesium, 0.392%phosphorus, 3.98% calcium and 1% lactose) was added to purified water toadjust the concentration of the whey protein to 12.4%. Then, 35%hydrochloric acid in an amount of 68 g was added to 4 kg this solutionand pH was adjusted to 2.95. This solution was passed through a columnfilled with am H⁺ type cation exchange resin AMBERLITE IR-120B (made byOrgano Company) in an amount of 350 ml at SV=10 h¹ to contact with eachother, and then passed through another column filled with an OH⁻ typeanion exchange resin AMBERLITE IRA-411 (made by Organo Company) in anamount of 700 ml at SV=5 h¹ to contact with each other. Then, thecolumns filled with resin were washed by purified water to recoversolids of the residual solution in them. The resultant solution of a pHof 3.50 containing a whey protein in an amount of 6.83 kg was recovered.

A 10% sodium hydroxide solution in an amount of 0.15 kg was added to therecovered solution, and pH was adjusted to 6.9. Ultrafiltration wascarried out through an ultrafiltration module SEP-1013 (made by AsahiKasei Company; having a fractional molecular weight of 3,000) todischarge lactose and inorganic matters on the membrane permeating side,thereby obtaining s desalted and lactose-removed whey protein solutionin an amount of 7.7 kg.

A 10% sodium hydroxide solution in an amount of 30 g was added to thisdesalted and lactose-removed whey protein solution, and pH was adjustedto 8.6. To this mixture, 4 g BIOPRASEsp-20 (made by Nagase Kagaku KogyoCompany), 2 g PTN6.0S (made by Novo Nordisk Company) and 4 g PROTEASE N"AMANO" (made by Amano Seiyaku Company) were added, and afterdecomposition at 50° C. for 14 hours, heated to 85° C. for ten minutesto inactivate the enzymes.

Then, this solution was subjected to an ultrafiltration through anultrafiltration module SEP-1013 (made by Asahi Kasei Company; afractional molecular weight of 3,000), and undissolved product remainingon the membrane was removed. The resultant filtrate was concentrated,and spray-dried by the conventional method, thereby obtaining about 256g spray-dried product of a low-phosphorus refined whey proteinhydrolysate.

The thus obtained powder was tested in accordance with theabove-mentioned test methods: the inorganic composition comprised 15.2%sodium, 0.22% potassium, 0.04% magnesium, 0.12% phosphorus and 0.19%calcium, with 0.48% chlorine, a lactose content of 0.26%, a fractionhaving a molecular weight of up to 1,200 accounting for 92.4%, a freeamino acid content of 5.4%, an antigenicity of up to 10⁻⁶ of that ofβ-lactoglobulin, and endotoxin of 5.15 EU/g per gram of dried wheyprotein hydrolysate.

Industrial Applicability

The low-phosphorus whey protein of the present invention is useful forincreasing nutritive value and enriching protein of various foodproducts.

The low-phosphorus purified whey protein of the present invention, ofwhich the phosphorus content is kept at a very low level, is useful infood manufacturing and medical areas as a protein nutritive source to beorally or directly administered to the stomach of the intestine for apatient suffering from renal failure or hyper-phosphatemia who isrequired to limit ingestion of phosphorus. It is also excellent innon-antigenicity and absorbency and is therefore applicable as a proteinnutritive source to be orally or directly administered to the stomach orthe intestine for patients suffering from allergy, decreased physicalfitness, gut immunity disease, allergic diarrhea, infants, and thosebefore and after operation. Because of the very low contents ofinorganic matters, lactose and endotoxin, it is applicable as a nitrogensource for an intravenous infusion or a peritoneum dialysis liquid.

We claim:
 1. A low-phosphorus whey protein comprising a phosphoruscontent of up to 0.15 mg per gram of protein.
 2. A method formanufacturing a low-phosphorus whey protein, which comprises the stepsof:(a) adjusting pH of a solution containing a whey protein to below 4;(b) contacting the solution obtained by step (a) with an H⁺ type cationexchange resin; and (c) sequentially contacting the solution obtained bystep (b) with an anion exchange resin to reduce the phosphorus contentper gram of protein to below 0.15 mg.
 3. The method according to claim2, wherein in step (c), the pH of the solution is adjusted to below 3.4. A low-phosphorus purified whey protein hydrolysate having thefollowing properties (1) to (6):(1) containing inorganic matters in thefollowing amounts per gram of protein:sodium: up to 20 mg, potassium: upto 20 mg, magnesium: up to 0.057 mg, phosphorus: up to 0.15 mg, calcium:up to 0.227 mg, and chlorine: up to 0.568 mg; (2) a lactose content ofup to 0.5% in weight; (3) a fraction having a molecular weight of up to1,200 at least 90% in weight; (4) a free amino acid content of up to 6%in weight; (5) an antigenicity, as measured by the enzyme linkedimmuno-sorbent assay, of up to 10⁻⁶ of antigenicity of β-lactoglobulin;and (6) an endotoxin content of up to 10 EU per gram of dried product.5. A method for manufacturing a low-phosphorus purified whey proteinhydrolysate as claimed in claim 4, which comprises the steps of:(a)adjusting pH of a solution containing a whey protein to below 4; (b)contacting the solution obtained by step (a) with an H⁺ type cationexchange resin; (c) sequentially contacting the solution obtained bystep (b) with an OH⁻ type anion exchange resin; (d) adjusting pH of thesolution obtained by step (c) to at least 5 and up to 10; (e) removinglactose from the solution obtained by step (d) through ultrafiltration;(f) adding an enzyme complex comprising two or more enzymes including aprotease derived from an animal and another protease isolated from aBacillus-genus microorganism, or an enzyme complex comprising three ormore enzymes including a protease derived from an animal, anotherprotease isolated from a Bacillus-genus microorganism and furtheranother protease to the solution obtained by step (e) in order toconduct enzymatic hydrolysis; (g) heating the solution obtained by step(f) to inactivate enzymes and simultaneously causing precipitation ofnon-reacting protein; and (h) removing fats and all precipitation fromthe solution obtained by step (g) through ultrafiltration.
 6. The methodaccording to claim 5, wherein in step (c), the pH of the solution isadjusted to below 3.